Not applicable.
Not applicable.
Phase contrast microscopy is of great importance in light microscopy for making biological structures visible which have no or only little amplitude contrast. The production of the phase contrast takes place in that different diffraction orders of the radiation diffracted at the object and already having a phase difference of xcfx80/2 due to the diffraction effect, have an additional phase shift of likewise xcfx80/2 impressed upon them, different for the different diffraction orders, by means of a phase plate. In light microscopy, phase contrast according to Zernike is produced in that an annular diaphragm is arranged in the pupil plane of the condenser, and a phase plate is arranged in the exit side focal plane of the microscope objective and impresses on the null beam, i.e., the light not diffracted at the object, a phase displacement which differs by xcfx80/2 from the phase displacement impressed on the higher diffraction orders. This method is not easily transferred to electron optics, since on one hand a submicroscopic annular diaphragm would be required in the illuminating system because of the small illuminating aperture, and on the other hand, no suitable support material is available which is completely transparent to electrons.
Various methods have already been proposed for the production of phase contrast in electron microscopy. One possibility is to use the phase-shifting effect of the aperture error of the objective in combination with a defocusing of the objective. However, the thereby produced phase contrast for a given defocus- and aperture error coefficient depends on the spatial frequency of the respective image information and is described by the so-called xe2x80x9cphase contrast transfer functionxe2x80x9d (PTCF). As a rule, however, such phase contrasts can only be made visible in a strong under-focus, in which the PCTF strongly oscillates, which then makes the interpretation of the image produced very difficult or even impossible.
A further possibility for the production of phase contrast in electron microscopy is described in an article by Boersch in Zeitschrift fxc3xcr Wissenschaften 2a, 1947, pages 615 ff. According to this, the phase displacement of the electron beam by external electrical and/or magnetic potentials or by the use of the internal potential on passage through a thin foil is used. In the latter principle, the phase plate consists of a thin carbon foil with a thickness of 20 nm with a central hole, through which the null beam can pass unaffected. The diffracted beams pass through the foil and undergo a phase displacement by xcfx80/2. This principle has substantial disadvantages, however. For one thing, the hole diameter of the central hole has to be of the order of magnitude of about 1 xcexcm, since the illuminating apertures and hence the expansion of the null beam in the focal plane is correspondingly small. The production and centering of such small holes relative to the electron beam is very problematic. Furthermore, the thickness of the foil can change greatly due to contamination, and the central hole for the null beam can quickly become overgrown due to contamination. The unavoidable electrical charging of the foil results in an additional anisotropic phase displacement, so that the total phase displacement produced becomes relatively uncontrollable. Finally, the higher diffraction orders of the radiation diffracted at the object, and already having a weaker intensity than the null beam, is additionally weakened in its intensity by the foil due to elastic and inelastic scattering, so that the contrast produced is correspondingly weak.
The first principle described in the abovementioned article, the use of the phase displacing effect of an external electrical potential, is further taken up in U.S. Pat. No. 5,814,815. An electrostatic single lens is used there for the displacement of the phase of the null beam with respect to the higher diffraction orders of the radiation diffracted at the object. Micromechanical manufacturing methods have to be used for the production of the single lens, because of the small spatial distances of the different diffraction orders. The construction of the annular electrostatic single lenses in a central position, and the supporting structures required for this, effect a stopping-out of the diffraction information. A further disadvantage is that the single lenses have to be of cantilevered construction and are therefore very difficult to implement. Further difficulties arise from the insulation of the middle electrode, since care must be strictly taken that no insulating materials, which could become charged by the electron beam, are present in the neighborhood of the beam. And here also the small central bore can quickly become polluted and overgrown due to contamination, as in the above-described alternative with the perforated foil.
The present invention therefore has as its object to provide an electron microscope with which the production of easily interpretable phase contrast images is possible and which has no components which are critical as regards production and for lasting retention of function. In particular, no cantilevered structures are to be required, i.e., structures held by thin supporting webs which bridge over the region of the beam cross section required for the production of the image.
The object of the invention is achieved by a transmission electron microscope with an optical axis, comprising an illuminating system for illuminating with an electron beam an object to be positioned in an object plane, and an objective for imaging the illuminated object; wherein the electron beam is split at the object into a null beam and higher diffraction orders; wherein the illuminating system produces an annular illuminating aperture in a Fourier transformed plane relative to the object plane; and wherein a phase-shifting element is arranged in a focal plane of the objective, remote from the object plane, and confers on the null beam a phase shift with respect to radiation diffracted at the object into higher diffraction orders; and wherein the phase-shifting element does not affect, or only slightly affects, the phase of the radiation diffracted at the object into higher diffraction orders and running closer in a radial direction to the optical axis than the null beam.
In the electron microscope according to the invention, the phase contrast is produced in a manner very similar to the production of phase contrast in light microscopy. The illuminating system of the electron microscope produces an annular illuminating aperture in a plane which is Fourier transformed with respect to the object plane to be imaged. As in phase contrast microscopy, the illumination of the object to be imaged consequently takes place with a beam of hollow conical shape. In the plane which is Fourier transformed with respect to the object plane or a plane conjugate thereto, a phase-shifting element is arranged which confers on the radiation undiffracted at the object, i.e., the null beam, a phase displacement with respect to the radiation diffracted at the object in higher diffraction orders. At the same time, the phase-shifting element permits the phase to be unaffected, or only slightly affected, of the radiation diffracted at the object at higher diffraction orders and running closer to the optical axis in the radial direction than the null beam.
In a transmission electron microscope according to the invention, as in phase contrast n light microscopy, a phase displacement is conferred by the phase-shifting element on the radiation undiffracted at the object with respect to the radiation diffracted at the object. The radiation diffracted at the object in higher orders of diffraction and running, in the plane of the phase-shifting element, closer to the optical axis in the radial direction than the radiation undiffracted at the object, is on the other hand not affected by the phase-shifting element. A corresponding phase-shifting element can therefore be constituted of annular shape with a central aperture. Such an annular phase-shifting element can consequently be mounted at its outer circumference, so that no cantilevered or nearly cantilevered structures are required. It even provides electron-optical advantages when the phase-shifting element or a retaining structure of the phase-shifting element stops-out the higher diffraction orders situated further from the optical axis in the radial direction with respect to the null beam. The negative effects of the off-axis aberrations of the objective are thereby reduced.
Furthermore, the phase-shifting element not only affects the phase of the null beam but also simultaneously weakens the intensity of the null beam due to a corresponding absorption. An overall improvement of contrast is achieved by the intensity matching which can be thereby attained between the null beam and the higher diffraction orders. A very stable construction of the phase-shifting element can be implemented by a combination of the phase-shifting element with an aperture diaphragm. The radiation of higher order diffracted toward the optical axis can pass unhindered through the central aperture of the phase-shifting element, while the radiation diffracted away in a radial direction from the optical axis is stopped-out. However, information is not lost by this stopping-out, since the illuminating beam rotated by 180xc2x0 relative to the optical axis contains the information complementary to the stopped-out diffraction orders.
A corresponding phase-shifting element is technologically simple to implement. The information-carrying diffracted beam of higher order does not undergo any negative effects, such as an attenuation or an additional phase shift due to its unhindered passage through the phase-shifting element, due either to the construction of the phase-shifting element or to its support. Moreover, no radiation diffracted in given spatial directions is completely stopped-out by support structures. The corresponding phase-shifting element can on the contrary be constituted rotationally symmetrical with respect to the optical axis.
Furthermore, small holes in the phase-shifting element, through which the primary beam has to pass, are avoided. Negative effects of contamination effects in addition arising with small holes are thereby substantially excluded. And since only information of null diffraction order, carrying no information about the object, passes through the phase-shifting element, variations of the phase shift due to local thickness fluctuations of the phase-shifting element are statistically equalized.
In an embodiment of the invention, the phase-shifting element is constituted as an annular electrode, whose electrostatic potential is variable.
In an alternative embodiment of the invention, the phase-shifting element is constituted as an annular foil, which is received on a heavy support. Both the annular foil and the heavy support respectively have an aperture directed perpendicularly of the optical axis, the diameter of the aperture in the annular foil being smaller than the aperture diameter of the heavy support. The heavy support can then serve at the same time as an aperture diaphragm for stopping-out the higher diffraction orders diffracted away from the optical axis.
A deflecting system is preferably provided in a plane conjugate to the object plane in order to produce the annular illuminating aperture. In this embodiment, the annular illuminating aperture is produced sequentially in time by variation of the deflection angle.
An alternative production of the annular illuminating aperture is also possible by means of a corresponding diaphragm with a central shadowing in the illuminating beam path. Furthermore, it is possible, particularly with thermal emitters as the electron source, to image the under-heated cathode image (hollow beam), which already has an annular emission distribution with a weak central maximum, in the front focal plane of the condenser-objective-single field lens, and to stop-out the central emission spot, in order to produce the annular illuminating aperture.